Processing milk at different temperatures has an impact on the physicochemical properties of the milk fat globule membrane (MFGM) which affect the surface properties of fat globules due to changes in the distribution and concentration of intrinsic proteins as well as others interacting as a consequence of the temperature. The aim of this study was to analyze the change in surface properties, such as zeta-potential and size distribution during different heat treatments and correlate it to shifts in proteins identified by 2D electrophoresis and a proteomic approach by tandem mass spectrometry. A pilot-scale study was performed using 5 conditions: no heating (raw), cooling at 4°C, batch (63°C for 30 min), high temperature-short time (HTST, 72°C, 16 s) and ultra-high temperature pasteurization (UHT, 121°C, 3 min). We identified 115 proteins classified in 12 families based on their function. The thermal treatments changed not only the native distribution of MFGM identified but also increased the adsorption and aggregation, especially of soluble serum proteins like α-lactalbumin and β-lactoglobulin as determined in 2D gel electrophoresis (Table 1). The change in surface properties and charge distribution was a direct consequence of thermal denaturation that favors intermolecular disulfide bonds of proteins at the MFGM surface since the formation of SS interaction was proven to correlate with heat treatment of milk. Among the pasteurization treatments, UHT resulted in the highest variation on surface charge and loss of proteins such as butyrophilin compared with HTST processing which had less protein denaturation. This promises to deliver MFGM isolates that maintain nutritional and functional activities when they are included in food products. Table 1. Protein distribution on the MFGM and relative abundance after different heat treatments

Family and Protein	Process
	Raw	4 °C	Batch	HTST	UHT
Lipid transport
Butyrophilin	5.5	4.4	3.8	1.8	1.0
XDH/XO	1.7	1.7	1.0	1.3	1.2
Milk proteins
αS1-Casein	5.6	4.8	2.1	2.5	1.0
αS2-Casein	1.0	1.6	2.8	1.4	6.3
κ-Casein	1.4	1.0	1.2	1.1
β-Lactoglobulin	1.8	1.2	10.3	10.2	20.5
α-Lactalbumin	1.6	1.7	2.4	2.0	3.8
Immunity
Lactadherin	6.7	9.1	7.0	8.9	1.0
Mucin 15	1.3	1.2	1.0	1.1	1.5
Mucin 1	1.2	1.0	1.3	1.0
Serum albumin	8.9	11.5	11.7	9.7	1.0